The regulation of activated transcription in eukaryotic cells involves an exquisite network of sequence-specific DNA-protein interactions working in concert with the timely recruitment of a multitude of coactivator proteins to modify chromatin structure and communicate with the preinitiation complex. HMGB1 protein exhibits context-dependent regulatory properties in that it can act as a coactivator (nuclear hormone receptors) or as a general repressor (on TBP). We have found that HMGB1 interaction with the estrogen receptor (ER)-DNA interactions broadly expands the spectrum of ER binding sites and strongly enhances their binding affinity to imperfect estrogen response elements (EREs), ERE half-sites (cHEREs), EREs with various spacers (cEREn), direct repeats, everted repeats and well-separated inverted repeats. Preliminary data also show that HMGB1 strongly enhances ER binding to cERE or cHERE to comparable levels within a phased nucleosome by a nonenzymatic mechanism. In addition, we have initiated transient transfection studies using luciferase reporter gene assays to begin to compare the activity of different EREs in activate transcription. These findings, together with recent findings from ChIP-chip experiments and genomic searches, bring into question the current paradigm for ER binding and activity. It also appears that the C-terminal extension (CTE) in ER may play a vital role in ER interactions with its response element. We shall use the very sensitive hydroxyl footprinting procedure to investigate whether the ER-CTE is involved in the DNA interaction, which would further change the current model for ER binding. In addition, we will investigate more systematically the role of HMGB1 in acting as a chromatin remodeling complex (CRC) in a phased nucleosome that contains a spectrum of single and multiple EREs. The remodeling activity of HMGB1 will be compared to the activity of the established ATP-dependent SWI/SNF CRC, in addition to determining if these two very different CRCs act independently or cooperatively. Chromatin immunoprecipitation experiments will determine if HMGB1 colocalizes with ER in EREs of estrogen-responsive genes in MCF-7 cells. Luciferase reporter genes assays will be used to compare the relative transcriptional activity of a spectrum of ER binding sites. PUBLIC HEALTH RELEVANCE: Our findings on estrogen receptors will lead to a deeper understanding of the mechanism by which estrogen affects normal physiology and development, in addition to the role the estrogen receptors play in the development of major diseases, including cancer, atherosclerosis, osteoporosis and dementia. [unreadable] [unreadable] [unreadable]